Microfluidic analysis system

ABSTRACT

A microfluidic analysis system (1) performs polymerase chain reaction (PCR) analysis on a bio sample. In a centrifuge (6) the sample is separated into DNA and RNA constituents. The vortex is created by opposing flow of a silicon oil primary carrier fluid effecting circulation by viscous drag. The bio sample exits the centrifuge enveloped in the primary carrier fluid. This is pumped by a flow controller (7) to a thermal stage (9). The thermal stage (9) has a number of microfluidic devices (70) each having thermal zones (71, 72, 73) in which the bio sample is heated or cooled by heat conduction to/from a thermal carrier fluid and the primary carrier fluid. Thus, the carrier fluids envelope the sample, control its flowrate, and control its temperature without need for moving parts at the micro scale.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/278,894, filed Sep. 28, 2016, which is a divisional of U.S.application Ser. No. 12/617,286, filed Nov. 12, 2009, which is acontinuation of U.S. application Ser. No. 11/366,524, filed Mar. 3,2006, which is a continuation of PCT/IE2004/000115, filed Sep. 6, 2004and published in English, claiming the priorities of U.S. ApplicationNos. 60/500,344 and 60/500,345, both filed on Sep. 5, 2003, each ofwhich are incorporated by reference in their entireties herein.

FIELD OF THE INVENTION

The invention relates to analysis systems for analysis such asPolymerase Chain Reaction (PCR) analysis to detect the population ofrare mutated cells in a sample of bodily fluid and/or tissue.

PRIOR ART DISCUSSION

It is known for at least the past decade that cancers have a geneticcause. With the emergence of fast methods of sequencing and thepublication of the human genome, the motivation and methods areavailable to find the genetic causes, both germline and somatic, of themost prevalent cancers. Contemporary oncological research suggests thatthere is a sequence of mutations that must occur for a cancer to belife-threatening, called the multistage model. Cancer could therefore bediagnosed earlier by detecting these genetic markers thereby increasingthe probability of cure. However, even with refining of the sample, thetarget cells and their DNA are still usually very rare, perhaps one partin 10⁶. The analysis system must therefore be able to perform veryeffective amplification.

There are several methods of attempting to identify rare cells in asample of bio-fluid. A common method is to probe the sample using knowngenetic markers, the markers being specific to the type of mutationbeing sought, and then amplify the targets in the sample. If themutations or chromosomal aberations are present then the amplificationcan be detected, usually using optical techniques.

It is also possible, depending on the amplification used, to use thePolymerase Chain Reaction (PCR) to detect the number of mutated cells inthe original sample: a number important as firstly, it can be linked tothe progress of the cancer and secondly, it provides a quantitativemeasure with which to diagnose remission. PCR is the enzyme-catalyzedreaction used to amplify the sample. It entails taking a small quantityof DNA or RNA and producing many identical copies of it in vitro. Asystem to achieve a. PCR is to process the samples by thermally cyclingthem is described in U.S. Pat. No. 5,270,183. However, this apparentlyinvolves a risk of sample contamination by surfaces in the temperaturezones and other channels. Also, U.S. Pat. No. 6,306,590 describes amethod of performing a PCR in a microfluidic device, in which a channelheats, and then cools PCR reactants cyclically. U.S. Pat. No. 6,670,153also describes use of a microfluidic device for PCR.

The invention is directed towards providing an improved microfluidicanalysis system for applications such as the above.

SUMMARY OF THE INVENTION

According to the invention, there is provided a biological sampleanalysis system comprising:

a carrier fluid;

a sample supply;

a sample preparation stage for providing a flow of sample enveloped in aprimary carrier fluid;

at least one analysis stage for performing analysis of the sample whilecontrolling flow of the sample while enveloped within the primarycarrier fluid without the sample contacting a solid surface; and acontroller for controlling the system.

In one embodiment, the analysis stages comprise a thermal cycling stageand an optical detection stage for performance of a polymerise chainreaction.

In another embodiment, the sample preparation stage comprises acentrifuge for separation of samples from an input fluid and forintroduction of the samples to the primary carrier fluid.

In a further embodiment, the centrifuge comprises a pair of opposedprimary carrier fluid channels on either side of a vortex chamber,whereby flow of primary carrier fluid in said channels causescentrifuging of sample in the vortex chamber and flow of sample from thechamber into said channels.

In one embodiment, contact between the sample and the vortex chambersurface is avoided by wrapping the sample in an initial carrier fluidwithin the chamber.

In another embodiment, the controller directs separation in thecentrifuge either radially or axially due to gravity according to natureof the input fluid such as blood containing the sample.

In a further embodiment, the primary carrier fluid velocity is in therange of 1 m/s to 20 m/s.

In one embodiment, the thermal cycling stage comprises a microfluidicthermal device comprising a thermal zone comprising a sample inlet forflow of sample through a sample channel while enveloped in the primarycarrier fluid, and a thermal carrier inlet for flow of a thermal carrierfluid to heat or cool the sample by heat conduction through the primarycarrier fluid.

In another embodiment, the microfluidic thermal device thermal zonefurther comprises separate sample and thermal outlets positioned toallow flow of thermal carrier fluid into and out of contact with theprimary carrier fluid.

In a further embodiment, there is at least one pair of opposed thermalcarrier inlet/outlet pairs on opposed sides of a sample channel.

In one embodiment, the thermal cycling stage comprises a plurality ofthermal zones.

In one embodiment, the microfluidic thermal device comprises a pluralityof thermal zones in series.

In another embodiment, the thermal cycling stage comprises a pluralityof microfluidic thermal devices in series.

In a further embodiment, the microfluidic thermal device comprises aclosed sample channel for re-circulation of sample with successiveheating or cooling in successive thermal zones.

In one embodiment, the controller directs flow of the thermal andprimary carrier fluids to control flowrate of sample by envelopingwithin the primary carrier fluid and by viscous drag between the thermalcarrier fluid and the primary carrier fluid.

In another embodiment, the primary carrier fluid is biologicallynon-reactive.

In a further embodiment, the primary carrier fluid is a silicone oil.

In one embodiment, the thermal carrier fluid is biologicallynon-reactive.

In another embodiment, the thermal carrier fluid is a silicone oil.

In a further embodiment, the temperatures and flowrates of the carrierfluids are controlled to achieve a temperature ramping gradient of 17°C./sec to 25° C./sec.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more clearly understood from the followingdescription of some embodiments thereof, given by way of example onlywith reference to the accompanying drawings in which:

FIG. 1 is a diagram of an analysis system of the invention.

FIG. 2 is a diagrammatic plan view of a centrifuge of the system, andFIG. 3 is a simulation diagram showing centrifuging;

FIG. 4 is a perspective view of the main body of a microfluidic beaterof the system;

FIG. 5 is a prediction velocity and temperature plot along a thermalstage of the heater;

FIG. 6 is a centre line temperature profile in the flow directionshowing fast response of same in the heated zone; and

FIG. 7 is a plan view of an alternative microfluidic heater.

DETAILED DESCRIPTION OF THE INVENTION Description of the Embodiments

Referring to FIG. 1 an analysis system 1 comprises a controller 2 whichinterfaces with various stages. A carrier fluid supply 4 deliverscarrier fluid to a macro pump 5 which delivers it at a high flowrate toa sample preparation stage 6. The latter also receives a bio-fluidsample, and centrifuges the sample in a vortex created by carrier fluidflow, as described in more detail below. Reactants are supplied by asupply 8 to a flow controller 7 which delivers streams of separated DNAwith reactants enveloped in carrier fluid to a thermal cycling stage 9.The DNA is amplified in the stage 9 and optically detected by adetection stage 10. Throughout the process the samples are enveloped ina biologically non-reactive carrier fluid such as silicone oil. Thisavoids risk of contamination from residual molecules on system channelsurfaces.

Referring to FIG. 2 a centrifuge device 20 of the sample preparationstage 6 is illustrated diagrammatically. It comprises opposed carriersupply lines 21 and 22 and a central vortex chamber 23 having a sampleinlet out of the plane of the page. The centrifuge 20 operates byprimary carrier fluid in the channels 21 and 22 driving sample fluid inthe chamber 23 into a vortex via viscous forces at the interface betweenthe two fluids. In this embodiment, the carrier fluid is silicone oilmixed to be neutrally buoyant with the sample.

The vortex, or centrifuge, is thus established without any mechanicalmoving parts. The carrier fluid drives a vortex of the sample to becentrifuged thereby avoiding the very many difficulties of designing andoperating moving parts at the micro scale, particularly at highrotational speeds. FIG. 3 illustrates the centrifuging activity, thegreater density of dots indicating higher flow velocities. The left-handscale shows the velocity range of 1 m/s to 20 m/s. The sample is wrappedin an initial volume of carrier fluid within the chamber 23 to preventsurface contamination.

This achieves a continuous throughput micro-centrifuging to suitablyextract DNA and RNA from cellular material. The bio-fluid is centrifugedresulting in DNA and other bio-molecules of interest accumulating at thebottom of the chamber, thereby providing an efficient and simple methodof manipulating micron and sub-micron quantities of bio-fluid. The DNAand RNA are separated due to the greater weight and viscous resistanceof the DNA. Numerical simulations (FIG. 3) of the flow show thattangential velocities of up to 10 ms⁻¹ are generated towards the edge ofthe vortex core. Calculations reveal this to be equivalent to arotational speed of almost 20,000 rpm or 2,000 g in terms of acentrifugal force. In order to achieve these levels of centrifugalforce, the carrier fluid is pumped at speeds of 5 ms⁻¹ through thesystem. In general, the desired carrier fluid speed is 1 m/s to 20 m/s.The device has further potential to be miniaturized to centrifuge at upto 200,000 g, as these levels of force are necessary for efficientseparation of RNA and other smaller cellular constituents andbio-molecules.

Overall, the continuous throughput centrifuge offers many benefits overconventional technology. The device may also function as a fluid mixingdevice by reversing the flow path of one of the carrier fluid, if suchis desired for an application. It is modular in nature, meaning two ormore systems can be placed together in any configuration and run by thesame control and power source system. The centrifuge 20 has no movingparts thereby allowing excellent reliability compared with a systemhaving moving pans. An important consequence of this feature is thatmanufacturing this device at the micro-scale using current siliconprocessing or micro-machining is readily achievable.

Referring to FIG. 4 a microfluidic thermal device 51 of the stage 9 isshown. It comprises three successive thermal zones 52, 53, and 54. Eachzone comprises a sample inlet 60 and an outlet 61 for flow of the biosample in the primary carrier fluid. There are also a pair of thermalcarrier inlets 65 and 66, and a pair of thermal carrier outlets 67 and68 for each of the three zones. This drawing shows only the main body,there also being top and bottom sealing transparent plates.

The bio sample which enters the sample inlet 60 of each stage isenveloped and conveyed by the carrier fluid henceforth called the“primary carrier fluid”. Thermal carrier fluid is delivered at theinlets 65 and 66 to heat or cool the bio sample via the primary carrierfluid.

As the sample remains in a low shear rate region of the flow, masstransport by diffusion of sample species is kept to a minimum. The lowshear region reduces damage by shear to macro molecules that may becarried by the bio sample. The arrangement of a number (in this casethree) of thermal zones in series offers advantages to applications suchas the polymerase chain reaction (PCR) where rapid and numerous thermalcycles lead to dramatic amplification of a DNA template strand.

The device 51 also acts as an ejector pump, in which the velocity andhence the residency time of the sample is controlled by controllingvelocity of one or both of the carriers fluids. The carrier flowparameters determine how long the sample remains at the set temperaturein each zone. This is often important, as chemical reactions requireparticular times for completion. The device 51 can therefore be tuned tothe required residency times and ramp rates by controlling the carriervelocity.

Referring to FIG. 5 a predicted velocity contour map at the mid-heightplane of a zone channel is shown. Carrier fluid enters through thechannels at the top and bottom left of the image and exits through thechannels at the top and bottom right of the image. The sample fluidenters and exits through the central channel. The different shadings ofthis map indicate the velocities, the range being 0.01 m/s to 0.1 m/s.

In one example, sample fluid enters through the central channel at theleft of the image at a temperature of 50° C. and is heated to 70° C. bythe thermal carrier fluid.

FIG. 6 shows a temperature profile along a longitudinal centerline of athermal zone. A target temperature of 342K is achieved within anextremely short distance from entrance, achieving an excellenttemperature ramp rate of 20° C/sec over a distance of 0.05 m. Ingeneral, a ramping of 17° C./sec to 25° C./sec is desirable for manyapplications.

The following table sets out parameters for one example. A silicone oil,density matched to the density of the bio sample, is used for both ofthe carrier fluids.

TABLE 2 Boundary Conditions and Fluid Properties Overall ChannelDimensions 5 mm × 5 mm × 200 mm Wall Boundary Condition outside ofAdiabatic carrier flow interaction zones Heat Transfer Carrier FluidInlet 70° C., 90° C., 110° C. temperature for each zone Sample/TransferCarrier Inlet Pressure   0 Pa Heat Transfer Carrier Fluid Inlet Pressure0.2 Pa Sample/Transport Carrier Outlet Pressure 1.9 Pa Heat TransferCarrier Fluid Outlet Pressure 1.7 Pa Mass Diffusivity 1.3 E−12 m²/sApproximate Temperature Gradient in 20° C./sec Zones

Referring to FIG. 7, another microfluidic thermal device, 70, is shown.There are again three thermal zones, however in this case on a generallyrectangular closed circuit, with zones 71, 72, and 73. The zones 71 and73 are on one side and there is only a single zone, 72, on the otherside. The thermal carrier fluid is silicone oil, as is the primarycarrier fluid. The thermal carrier fluid for the zone 71 is at 68° C.,to ramp up the bio sample to this temperature during residency in thiszone. The zones 72 and 73 provide outlet temperatures of 95° C. and 72°C. respectively.

The optical detection stage 10 is positioned over the microfluidicdevice 70 to analyze the sample. The silicone oil is sufficientlytransparent to detect the fluorescently tagged molecules.

It will be appreciated that the invention achieves comprehensive controlover bio sample flowrate and temperature, with no risk of contaminationfrom device surfaces. The invention also achieves integrated pumping andthermal cycling of the sample without moving parts at the microscale.There are very high throughputs as measured by processing time for onesample.

The system is expected to have a low cost and high reliability due tothe absence of micro scale moving parts. The system also allowsindependent control and variation of all PCR parameters for processoptimization.

The invention is not limited to the embodiments described but may bevaried in construction and detail.

What is claimed is:
 1. A system for analyzing a biological sample, thesystem comprising: a sample preparation subsystem comprising: an firstfluid supply line configured to supply a flow of biological sample fluidcontaining target nucleic acid; and an second fluid supply lineconfigured to supply a flow of carrier fluid immiscible with thebiological sample fluid, wherein the sample preparation device isconfigured to: separate from the biological sample fluid strands oftarget nucleic acid, and deliver the separated strands of target nucleicacid enveloped in carrier fluid; a thermal cycling subsystem configuredto receive the separated strands of target nucleic acid enveloped incarrier fluid and to amplify the strands of target nucleic acid tocreate amplified product target nucleic acid; and a detection subsystemconfigured to detect signal from the amplified product target nucleicacid.
 2. The system of claim 1, wherein the detection subsystem isconfigured to detect the signal from the amplified product of targetnucleic acid in a state of a flow of the amplified product targetnucleic acid enveloped in carrier fluid.
 3. The system of claim 1,further comprising a controller configured to control velocity of a flowof the amplified product of target nucleic acid enveloped in carrierfluid in the detection subsystem.
 4. The system of claim 1, wherein thedetection subsystem is configured to detect fluorescence.
 5. A methodfor analyzing a biological sample, the method comprising: in a samplepreparation stage: flowing a supply of biological sample fluidcontaining target nucleic acid, flowing a supply of carrier fluidimmiscible with the biological sample fluid, separating strands oftarget nucleic acid in the biological sample fluid, and delivering theseparated strands of target nucleic acid enveloped in a carrier fluid toa thermal cycling stage; in the thermal cycling stage, amplifying thestrands of target nucleic acid to create amplified product targetnucleic acid; and in a detection stage, detecting signal from theamplified product target nucleic acid.
 6. The method of claim 5, whereinthe carrier fluid is oil.
 7. The method of claim 5, wherein flowing thesupply of carrier fluid comprises pumping the carrier fluid.
 8. Themethod of claim 5, further comprising centrifuging the biological samplefluid to cause the separating of the strands of target nucleic acid. 9.The method of claim 5, wherein the separated strands of target nucleicacid are on an order of microns or sub-microns.
 10. The method of claim5, wherein the detecting occurs while flowing the amplified producttarget nucleic acid enveloped in carrier fluid.
 11. The method of claim5, further comprising, in the detection stage, a controlling a flow ofamplified product target nucleic acid enveloped in carrier fluid duringdetecting.
 12. The method of claim 5, wherein the detecting signal fromthe amplified product target nucleic acid comprises detectingfluorescence emitted from the amplified product target nucleic acid. 13.The method of claim 5, further comprising a controlling temperaturecycling of the thermal cycling subsystem to perform the amplifying ofthe strands of target nucleic acid.
 14. The method of claim 5, whereinthe detecting of signal from the amplified product target nucleic acidoccurs during the amplifying in the thermal cycling stage.
 15. Themethod of claim 5, further comprising, in the sample preparation stage,accumulating the separated strands of target nucleic acid in a chamber.16. A method for analyzing rare mutated cells from a sample of bodilyfluid or tissue using polymerase chain reaction (PCR), the methodcomprising: forming droplets enveloped in an immiscible carrier fluid,wherein the droplets are configured to capture nucleic acid moleculesfrom at least some rare mutated cells; extracting the nucleic acidmolecules from the at least some rare mutated cells; amplifying thenucleic acid molecules from the at least some rare mutated cells withPCR; analyzing the nucleic acid molecules from the at least some raremutated cells, wherein the analysis is configured to target cells thatoccur about one part in
 106. 17. The method of claim 16, whereinamplifying the nucleic acid molecules further includes continuouslyflowing the droplets enveloped in the immiscible carrier fluid through aplurality of thermal zones.
 18. The method of claim 16, whereinamplifying and analyzing the nucleic acid molecules is performed in amicrofluidic device.
 19. The method of claim 16, wherein a fluid streamdelivers one or more reactants for amplifying the nucleic acidmolecules.